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N. A. Finch, P. J. Linser, J. D. Ochrietor; The C-Terminal Region of the Basigin Transmembrane Domain Interacts With MCT1. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3070.
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It has been demonstrated that monocarboxylate transporter-1 (MCT1) interacts with Basigin using immunoprecipitation and fluorescence resonance energy transfer (FRET) techniques. It has been hypothesized that these two membrane proteins interact via the transmembrane domain of Basigin. We therefore sought to determine which amino acids are necessary for this interaction.
A full-length 6X-histidine-tagged transmembrane protein (24 amino acids), as well as 6X-histidine-tagged deletion mutants were generated using the pET102/D vector (Invitrogen). The domain was truncated from both the N- and C-termini. ELISA analyses, in which endogenous MCT1 was captured and probed with the full-length Basigin transmembrane domain or a deletion mutant, were performed.
We found that the probe containing the entire transmembrane domain of Basigin (amino acids 1 to 24) did interact with MCT1. An interaction was also observed when a probe containing amino acids 13 to 24 of the Basigin transmembrane domain was used. A comparable interaction was observed when amino acids 19 to 24 were used as a probe, but no signal was observed when amino acids 13 to 18 were used.
The results indicate that the last six C-terminal amino acids of the Basigin transmembrane domain bind to MCT1. Future studies, including site directed mutagenesis will be aimed at determining the contribution of each of the six amino acids to this binding interaction.
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